1. Field of the Invention
The present invention is concerned with peptide reagents and compositions thereof which reduce blood clot formation.
2. Description of the Related Art
Blood clotting relies upon a series or cascade of activating reactions to produce the ultimate fibrin clot. The cascade leading to fibrin formation may be triggered initially in two different ways--by contact with abnormal surfaces (the "intrinsic pathway") or by traumatization of blood vessels which causes secretion of the lipoprotein known as "tissue factor" or TF (the "extrinsic pathway"). The present invention is primarily concerned with the extrinsic blood clotting pathway.
TF is an integral membrane protein which appears on many cell types. However, cells which constitutively express TF, for example the muscle cells of vessels intima, are not normally exposed to blood (see Edgington et al., Thromb. Haemostas. 66(1): 67-69 (1991)). Thus initiation of the extrinsic blood clotting pathway appears to require either the disruption of blood vessel walls (see Almus et al., Blood 76: 354-360 (1990)) and/or activation of endothelial cells or monocytes to express TF (see Edwards et al., Blood 54: 359-370 (1979) and Bevilaqua et al., PNAS USA 83: 4533-4537 (1986)). Disruption of the blood vessel wall may occur due to fissuring of an atherosclerotic plaque which exposes tissue macrophages and smooth muscle cells to the blood (see Wilcox et al., PNAS USA 86: 2839-2843 (1989)). TF may also be exposed following injury to blood vessels during thrombolytic therapy, surgery for grafting, mechanical restoration of vessel patency or other similar techniques. On the other hand, TF expression in endothelial cells or in monocytes may be induced during sepsis due to production of tumour necrosis factor-.alpha. or interleukin-1 (see Edwards et al., supra and Gregory et al., J. Clin. Invest. 76: 2440-2445 (1985)).
The serine protease Factor VIIa (FVIIa) is involved in the extrinsic blood clotting pathway. FVIIa is formed by proteolysis from its inactive pro-enzyme Factor VII (FVII) by other participants in the blood clotting process, including Factor Xa, Factor XIIa, Factor IXa or thrombin. Activation of FVII to FVIIa has been reported to be markedly enhanced when FVII is bound to its co-factor tissue factor (TF) (see Nemerson, Semin. Hematol. 29(3): 170-176 (1992)). Yamamoto et al. have also suggested that conversion of FVII to FVIIa may be autocatalytic (see J. Biol. Chem. 267(27): 19089-19094 (1992)).
FVIIa forms a complex with TF in the presence of calcium ions and the FVIIa/TF complex catalyses the conversion of Factor X to its active form, Factor Xa, in the next step of the blood clotting process via the extrinsic pathway.
The structure of FVII has been investigated and the cDNA sequence was reported by Hagen et al. in PNAS USA 83: 2412-2416 (1986). FVII is a vitamin K dependent protein and, by analogy to other vitamin K dependent proteins, a putative .gamma.-carboxyglutamic acid (Gla) domain has been identified at the amino terminal. It was predicted, again by analogy to the other vitamin K dependent proteins, that the Gla domain was required for binding to TF (see Hagen et al., supra). The Gla domain is followed by two potential growth factor (GF) domains.
However, the literature has not suggested any function for the GF domains.
Activation of the extrinsic pathway for blood clot formation has been suggested as the primary event leading to fibrin formation (see Weiss et al., Blood 71: 629-635 (1988) and Weiss et al., Blood 73: 968-975 (1989)) and is thus of prime importance in the pathogenesis of arteriosclerotic lesions and in reocculusion and and restenosis following endarterectomy. However effective therapeutic agents able to intervene in the activation of this pathway are not available, despite demand (see Shepard, TIBTECH 9: 80-85 (1991)).
The present invention provides a novel peptide and analogues or salts thereof which inhibit the association of FVII or FVIIa with TF. Through the action of the peptides according to the invention, formation of the FVIIa/TF complex is limited and therefore activation of Factor X is reduced.
Certain peptides stated to be useful in blood clotting therapy are disclosed in WO-A-91/07432 of the Board of Regents, The University of Texas System. The peptides disclosed either occur in the region between the Gla and the first GF domain or in the catalytic domain of FVII or FVIIa. Although inhibition of FVIIa/TF complex formation is discussed, those peptides disclosed in WO-A-91/07432 which cause such an effect do so via inhibition of the Gla function. Such peptides are thus unspecific in their action since other physiological proteins have Gla domains, for example protein C which has close sequence homology to the Gla domain of FVII. Hence, the function of protein C would also be disturbed by peptides disclosed in WO-A-91/07432 in an undesirable way.
In WO-A-90/03390, Corvas Inc. suggest that certain peptides derived from the amino acid sequence of FVII (or FVIIa) might be useful in preventing the action of the fully formed FVIIa/TF complex. Two particular peptide sequences were disclosed in WO-A-90/03390 as being active in this respect. The sequence -VGHFGV- is based upon amino acids nos. 372-377 of FVII which are situated near the carboxy terminus. The other sequence is -SDHTGTKRSCR- which is located at amino acids nos. 103-113 of FVII and is part of the second GF domain. Corvas Inc indicate that these peptides, and analogues thereof, inhibit the cascade reaction initiated by the FVIIa/TF complex.
Furthermore, in Table 1, on page 14 of W090/03390, it is indicated that of the various regions of the second GF domain, only SDHTGTKRSC (103 to 112) was active and that the other regions, namely from amino acids 50 to 101 and 114 to 127, were totally inactive in inhibition of activation of Factor X by Factor VII and tissue factor.
However, we have shown that, contrary to the reported findings of Corvas Inc., the region SDHTGTKRSC from 103 to 112 is a rather poor inhibitor of the binding of Factor VII to tissue factor and that certain of the regions said to be inactive are, in fact, highly active.
Our co-pending International application PCT/GB94/01315 discloses peptide fragments from the region 91 to 104 of the Factor VII sequence which are particularly active as inhibitors of the binding of Factor VII to tissue factor (TF) and comparative results are provided for several of these as compared with fragments from neighbouring regions, including the above fragment of Corvas Inc. Although a Table was provided which identified fragments which had been made, two of the Figures included results for a compound identified as 98-102(C) which was not in the Table. The reference (C) meant that the compound, which has the amino acid sequence CEQYC (Cys-Glu-Gln-Tyr-Cys), was in a cyclic form in which the two C (cysteine) residues were joined by a disulphide bond. Although not stated, the result indicated by the Figure was that for a derivative in cyclic form having an N-terminal acetyl group and a C-terminal carboxamide group, namely, disulphide-cyclo-Ac-Cys-Glu-Gln-Tyr-Cys-NH.sub.2 !. On the other hand, the Table included a fragment identified as "98-102" which was, however, indicated to be AEQYV, an analogue of CEQYC in which the terminal cysteine residues were replaced by alanine and valine, and was thus not capable of forming a cyclic derivative. The inhibition of binding of Factor VII to tissue factor reported for the so-called 98-102(C) was negligible.